156 Chapter 8 of samples and make the process more efficient (Deurenberg et al. 2017; Quainoo et al. 2017). Moreover, the use of WGS data makes it not only possible to analyse acquired resistance genes but also to screen for alterations that may lead to hyperproduction of chromosomal-encoded genes, e.g., the chromosomal ampC in E. coli. Furthermore, the clonal relatedness of the strains can be analyzed using wgMLST (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016; Quainoo et al. 2017). This makes WGS a versatile method for the analysis of resistance mechanisms. A limitation in the present study could be the use of the whole genome coverage as a reference for the ePCN calculation. As coverage of plasmid-encoded DNA is included in this average coverage, this might result in an overestimation of actual chromosomal coverage, and a higher plasmid copy number may increase this effect. The coverage of household MLST genes was slightly lower than the overall genome coverage in all isolates, although the ratio did not differ substantially. To limit this effect, the median coverage could be used, for example, in PLACNET, PlasmidSpades, and PlasmidSeeker in which the hypothesis of copy number differences in chromosomal-encoded and plasmid-encoded DNA is used to assemble plasmids (Lanza et al. 2014; Antipov et al. 2016; Roosaare et al. 2018). In the calculation of the estimated plasmid copy number, it was assumed that there were no repeats of chromosomal-encoded genes present, so-called copy number variations (CNVs). These CNVs could lead to an overestimation of the actual chromosomal coverage. Although we cannot exclude that CNVs were present, we do not expect that this would have influenced our conclusion. The present study shows that an increased estimated plasmid copy number of blaCMY-2 carrying plasmids is associated with reduced susceptibility for CTX, CAZ, and TZP. It is uncertain whether this association can be generalized to other plasmid-encoded resistance genes, e.g., ESBL. If so, this could aid the translation of molecular data into clinically meaningful results. Further studies are warranted to analyse the association between ePCN and other plasmid-encoded resistance genes. Conclusion The present study applied sequencing depth to explore the association between MICs of beta-lactam antibiotics and an estimated plasmid copy number. The results show a significantly higher ePCN for the scaffolds containing a blaCMY-2 gene in the isolates with high MIC values for CTX, CAZ and TZP compared to the isolates with low MIC
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